A worldwide collection of Fusarium oxysporum f. sp. cubense was analyzed using anonymous, single-copy, restriction fragment length polymorphism (RFLP) loci. Several lines of evidence indicated that this pathogen has a clonal population structure. Of the 165 isolates examined, only 72 RFLP haplotypes were identified, and nearly half the isolates were represented by the five most common haplotypes. Individuals with identical haplotypes were geographically dispersed, and clone-corrected tests of gametic disequilibrium indicated significant nonrandom association among pairs of alleles for 34 of 36 loci tested. Parsimony analysis divided haplotypes into two major branches (bootstrap value = 99%) that together contained eight clades supported by significant bootstrap values. With the exception of two isolates, all isolates within a vegetative compatibility group were in the same clade and clonal lineage. Clonal lineages were defined by isolates having coefficients of similarity between 0.94 and 1.00. Ten clonal lineages were identified, and the two largest lineages had pantropical distribution. Minor lineages were found only in limited geographical regions. Isolates composing one lineage (FOC VII) may represent either an ancient genetic exchange between individuals in the two largest lineages or an ancestral group. The two largest lineages (FOC I and FOC II) and a lineage from East Africa (FOC V) are genetically distinct; each may have acquired the ability to be pathogenic on banana independently.